Recombinant Acinetobacter baumannii NADH-quinone oxidoreductase subunit A (nuoA)

What is NADH-quinone oxidoreductase subunit A (nuoA) in Acinetobacter baumannii?

The nuoA subunit, while small compared to other Complex I components, plays a critical role in maintaining the structural integrity of the complex and potentially contributes to the proton pumping machinery. Understanding its structure and function provides insights into A. baumannii's energy metabolism, which may be particularly important given this pathogen's ability to survive in diverse environments and its concerning antibiotic resistance profile .

Why is the study of recombinant nuoA important for understanding A. baumannii pathogenesis?

The study of recombinant nuoA is critical for elucidating A. baumannii's pathogenesis for several key reasons. First, energy metabolism is fundamental to bacterial survival during infection, and as a component of Complex I, nuoA contributes to the bacterium's ability to generate energy under the varying conditions encountered within a host. Second, recombinant expression allows researchers to produce sufficient quantities of this membrane protein for detailed structural and functional analyses that would be difficult to perform with native protein complexes. Third, genetic manipulation of recombinant nuoA enables structure-function studies through site-directed mutagenesis, providing insights into how specific amino acid residues contribute to protein function and potentially to pathogen survival .

Additionally, A. baumannii has been designated as a "red alert" human pathogen due to its extensive antibiotic resistance spectrum, which has increasingly become a cause for serious concern in both nosocomial and community-acquired infections . The bacterium's ability to rapidly acquire resistance genes has been demonstrated through genomic studies showing that resistant strains contain resistance "islands" with clusters of up to 45 resistance genes . Understanding the biology of essential components like nuoA may reveal new vulnerabilities that could be exploited for therapeutic intervention, particularly important as conventional antibiotic approaches become increasingly challenged by resistance mechanisms.

What expression systems are most effective for producing recombinant A. baumannii nuoA?

The selection of an expression system for recombinant A. baumannii nuoA production requires careful consideration of several factors due to its nature as a membrane protein. The following table outlines the most effective expression systems with their respective advantages and limitations:

When working with recombinant nucleic acid molecules for nuoA expression, researchers must adhere to established biosafety guidelines, such as the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules . These guidelines specify practices for constructing and handling recombinant nucleic acid molecules, synthetic nucleic acid molecules, and cells containing such molecules . Since A. baumannii is an opportunistic pathogen with clinical significance, appropriate biosafety measures must be implemented during research activities.

How can the functional integrity of recombinant nuoA be verified?

Verifying the functional integrity of recombinant nuoA presents unique challenges since nuoA is a subunit of a larger complex and may not exhibit catalytic activity in isolation. A comprehensive approach to verification includes:

First, structural integrity assessment through techniques such as circular dichroism spectroscopy to confirm proper secondary structure content, particularly the alpha-helical content expected in membrane proteins. Thermal stability assays using differential scanning fluorimetry can further evaluate protein folding quality. Second, membrane incorporation can be verified through sucrose gradient centrifugation of membrane fractions or through fluorescence microscopy if the protein is tagged with a fluorescent marker.

Third, interaction studies using pull-down assays, surface plasmon resonance, or crosslinking experiments can confirm whether recombinant nuoA properly interacts with other Complex I subunits. Fourth, when incorporated into the complete Complex I, NADH oxidation activity can be measured spectrophotometrically by monitoring the decrease in NADH absorbance at 340 nm . The NADH/NAD+ ratio is crucial for cellular energy metabolism, and proper functioning of NADH-oxidizing enzymes like Complex I is essential for maintaining this balance .

Finally, functional complementation experiments, where recombinant nuoA is expressed in A. baumannii strains with nuoA deletions, can demonstrate whether the recombinant protein can restore respiratory function in vivo, providing the most physiologically relevant verification of functional integrity.

How does the structure of nuoA contribute to proton translocation in the NADH-quinone oxidoreductase complex?

The nuoA subunit's contribution to proton translocation in NADH-quinone oxidoreductase complex involves both structural and functional roles within the membrane domain of Complex I. While high-resolution structural data specifically for A. baumannii nuoA is limited, insights from homologous systems suggest that nuoA contains multiple transmembrane helices that interact with other membrane subunits to form part of the proton translocation pathway. These helices likely contain conserved charged or polar residues that participate in proton transfer through the membrane.

The proton translocation mechanism in Complex I involves a conformational coupling between electron transfer in the hydrophilic domain and proton pumping in the membrane domain. This coupling must transmit energy over a considerable distance from the site of NADH oxidation to the membrane-embedded proton channels. NuoA, positioned within the membrane domain, may contribute to this mechanism by participating in conformational changes induced by electron transfer. Studies on NADH oxidation have demonstrated that modulation of NADH/NAD+ ratios can significantly affect cellular energy metabolism, highlighting the importance of proper coupling between electron transfer and proton translocation .

What challenges exist in purifying recombinant nuoA for structural studies?

Purification of recombinant nuoA for structural studies presents multiple significant challenges inherent to membrane protein biochemistry, requiring specialized approaches to overcome them. First, membrane protein overexpression often leads to toxicity in host cells, necessitating careful optimization of expression conditions. Reduced expression temperatures (16-20°C), lower inducer concentrations, and specialized expression hosts are typically required to balance protein production with host cell viability.

Second, extraction from membranes requires careful selection of detergents. Traditional detergents like DDM (n-dodecyl-β-D-maltoside) or LMNG (lauryl maltose neopentyl glycol) may be suitable starting points, but systematic screening is necessary to identify conditions that maintain nuoA's native fold. Newer approaches using styrene-maleic acid copolymers (SMALPs) allow extraction of membrane proteins with their surrounding lipid environment intact, potentially preserving native interactions.

Third, purification protocols must maintain protein stability throughout multiple chromatography steps. Inclusion of lipids in purification buffers, precise pH control, and addition of stabilizing agents may be necessary. Protein-specific factors also complicate purification—nuoA's small size and multiple transmembrane domains make it prone to aggregation when removed from its native lipid environment. Additionally, distinguishing between properly folded protein and misfolded aggregates requires careful quality control using techniques like size exclusion chromatography and dynamic light scattering.

Finally, obtaining sufficient quantities of purified protein for structural studies remains challenging. While X-ray crystallography traditionally requires milligram quantities of highly pure protein, newer approaches like cryo-electron microscopy may be more suitable for membrane proteins like nuoA, potentially requiring less material while providing valuable structural insights in a more native-like environment.

How do mutations in nuoA affect electron transport chain efficiency in A. baumannii?

Mutations in nuoA can significantly impact electron transport chain efficiency in A. baumannii through several mechanisms, with consequences for bacterial energy metabolism and potentially for pathogenesis. Primary effects include disrupted complex assembly, where mutations in key interface regions can prevent proper incorporation of nuoA into Complex I, leading to incomplete complex formation and reduced respiratory capacity. Structural perturbations may alter the positioning of transmembrane helices critical for proton translocation, uncoupling electron transfer from proton pumping and reducing energy conversion efficiency.

Studies on related NADH-oxidizing enzymes have demonstrated that even single amino acid substitutions can dramatically reduce enzymatic activity. For example, in NADH:flavin oxidoreductase from Aminobacter aminovorans, mutation of a conserved histidine residue (His140) to alanine reduced enzyme activity to less than 3% . While the specific molecular context differs between this enzyme and Complex I, this finding illustrates how sensitive NADH-oxidizing enzymes can be to mutations in key residues.

Secondary effects of nuoA mutations extend beyond Complex I itself. Reduced Complex I activity forces metabolic adaptations, potentially increasing reliance on alternative respiratory complexes or fermentative pathways. This metabolic rewiring can affect growth rates, particularly under oxygen limitation. Furthermore, altered electron flow may increase production of reactive oxygen species if electrons leak from the transport chain, potentially causing oxidative stress and activating stress response pathways. In the context of A. baumannii as an opportunistic pathogen with extensive antibiotic resistance , these metabolic shifts might influence susceptibility to antimicrobials or the bacterium's ability to persist in challenging host environments.

What methodological approaches can determine nuoA's interaction partners within the respiratory complex?

Determining nuoA's interaction partners within the respiratory complex requires a multi-faceted approach combining biochemical, biophysical, and genetic techniques to build a comprehensive interaction map. In vivo crosslinking with membrane-permeable crosslinkers followed by immunoprecipitation and mass spectrometry analysis can capture physiologically relevant interactions within intact bacterial cells. This approach preserves the native membrane environment and can identify both stable and transient interactions. Complementary to this, bacterial two-hybrid systems adapted for membrane proteins allow screening for protein-protein interactions in a cellular context, though care must be taken to ensure proper membrane insertion of fusion proteins.

For more detailed biophysical characterization, surface plasmon resonance (SPR) or microscale thermophoresis (MST) can quantify binding affinities between purified nuoA and potential partner proteins, providing kinetic and thermodynamic parameters of these interactions. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) offers insights into interaction interfaces by identifying regions protected from deuterium exchange upon complex formation. This technique is particularly valuable for membrane proteins where traditional structural biology approaches may be challenging.

Genetic approaches provide functional context for these interactions. Suppressor mutation analysis, where secondary mutations compensate for nuoA mutations, can reveal functional relationships between nuoA and other complex components. Similarly, genetic complementation tests with chimeric proteins can identify functionally important interaction domains. The integration of data from these diverse approaches allows researchers to build a detailed map of nuoA's interaction network and understand how these interactions contribute to the assembly and function of the NADH-quinone oxidoreductase complex in A. baumannii, an opportunistic pathogen of increasing clinical concern .

How does redox sensing influence nuoA expression and Complex I assembly in A. baumannii?

Redox sensing plays a crucial role in regulating nuoA expression and Complex I assembly in A. baumannii, creating a sophisticated feedback system that allows the bacterium to adapt its respiratory capacity to changing environmental conditions. The NADH/NAD+ ratio serves as a key redox indicator that influences this regulatory network . When this ratio increases, signaling excess reducing equivalents, A. baumannii likely responds by adjusting expression of respiratory chain components, including nuoA, to restore redox balance. This regulation involves multiple interconnected mechanisms operating at transcriptional, post-transcriptional, and post-translational levels.

At the transcriptional level, A. baumannii possesses redox-responsive transcription factors similar to those characterized in related bacteria, which may directly regulate nuoA expression. These factors likely sense redox status through iron-sulfur clusters, thiol-based switches, or direct interaction with NAD+/NADH. Post-transcriptionally, small regulatory RNAs whose expression is influenced by redox conditions may modulate nuoA mRNA stability or translation efficiency. At the protein level, the redox state affects chaperone activities critical for proper membrane protein folding and complex assembly. Oxidative stress can disrupt disulfide bond formation and protein folding, directly impacting Complex I assembly.

Furthermore, the cellular redox state influences lipid metabolism and membrane composition, which in turn affects the membrane environment where Complex I is assembled and functions. Experiments manipulating the NADH/NAD+ ratio have demonstrated significant effects on cellular energy metabolism , suggesting that maintaining appropriate redox balance is essential for proper respiratory chain function. For A. baumannii, an opportunistic pathogen associated with hospital-acquired infections , this redox-responsive regulation may be particularly important for adaptation to the various microenvironments encountered during infection, including oxygen-limited conditions and oxidative stress from host immune responses.

How can researchers distinguish between direct effects on nuoA function versus indirect metabolic consequences when analyzing mutant phenotypes?

Distinguishing direct effects on nuoA function from indirect metabolic consequences when analyzing mutant phenotypes requires a systematic experimental approach integrating multiple levels of analysis. A comprehensive strategy begins with precise genetic engineering using techniques like scarless mutagenesis to create point mutations in nuoA without polar effects on downstream genes. These mutations should target specific functional domains based on structural predictions and conservation analysis. Complementation studies, where wild-type nuoA is reintroduced into mutant strains, confirm phenotype specificity while ruling out secondary mutations elsewhere in the genome.

Biochemical assays provide direct functional measurements of Complex I activity. NADH oxidation rates in membrane preparations or with purified complexes directly assess electron transfer activity, while proton pumping measurements using pH-sensitive dyes in reconstituted proteoliposomes evaluate proton translocation efficiency. These assays should be performed under various substrate concentrations to generate full kinetic profiles, as alterations in Km values versus Vmax can suggest different types of functional impairment. Comparing activities of Complex I versus other respiratory chain components helps distinguish nuoA-specific defects from general respiratory dysfunction.

Metabolomic analysis provides crucial context for distinguishing direct from indirect effects. Targeted metabolomics focusing on central carbon metabolism intermediates, particularly NAD+/NADH ratios and other redox couples, can reveal compensatory metabolic shifts . Global metabolomics may identify unexpected metabolic adaptations. Time-course experiments following mutation induction help separate primary effects from secondary adaptations, as direct functional consequences typically appear immediately while compensatory responses develop over time.

Systems biology approaches complete the analysis framework. Transcriptomic profiling identifies activation of stress responses or metabolic rewiring, while proteomic analysis reveals changes in respiratory complex stoichiometry or stress response activation. Computational modeling integrating these multilevel datasets can predict metabolic flux redistributions and distinguish primary effects from downstream consequences, providing a comprehensive understanding of how nuoA mutations impact A. baumannii physiology.

What role does nuoA play in A. baumannii's adaptation to antibiotic stress and development of resistance?

The nuoA subunit plays multifaceted roles in A. baumannii's adaptation to antibiotic stress and potentially contributes to resistance development through several interconnected mechanisms. Primarily, as part of Complex I, nuoA contributes to maintaining the proton motive force (PMF), which is critical for energizing many antibiotic efflux pumps. Alterations in nuoA function can modulate PMF, potentially affecting the efficiency of these efflux systems that contribute significantly to A. baumannii's intrinsic antibiotic resistance . Furthermore, the energetic state of the cell, influenced by respiratory chain function, affects the bacterium's ability to repair damaged cellular components following antibiotic exposure.

Metabolically, nuoA's role in Complex I affects the NADH/NAD+ ratio, which has wide-ranging implications for bacterial physiology under antibiotic stress . This ratio influences the redox state of the cell and the activity of various metabolic pathways that may contribute to intrinsic resistance mechanisms. For instance, altered NADH/NAD+ ratios can affect the pentose phosphate pathway, influencing the production of reducing equivalents needed for detoxification of oxidative stress, which may be induced secondarily by some antibiotics.

Genetically, A. baumannii demonstrates remarkable genomic plasticity, as evidenced by the presence of resistance "islands" containing clusters of up to 45 resistance genes in virulent strains . While nuoA mutations themselves may not directly confer resistance, altered respiratory chain function may influence the selective pressures that drive the acquisition and maintenance of resistance determinants. Metabolic adaptations resulting from nuoA mutations could potentially create cellular environments that favor the functional integration of horizontally acquired resistance genes.

Experimentally, investigating nuoA's role in antibiotic resistance requires examining how nuoA mutations affect susceptibility profiles across different antibiotic classes, analyzing the expression of efflux systems in nuoA mutants, and measuring the acquisition rates of resistance determinants in strains with altered nuoA function. This multifaceted approach would provide insights into how respiratory chain components like nuoA contribute to A. baumannii's concerning antibiotic resistance profile .

What potential exists for developing small molecule inhibitors targeting nuoA as novel antimicrobials against A. baumannii?

The development of small molecule inhibitors targeting nuoA represents a promising but challenging approach for novel antimicrobials against A. baumannii. The strategic value of nuoA as a target stems from several key factors. First, as a component of the NADH-quinone oxidoreductase complex, nuoA contributes to a fundamental metabolic process—cellular respiration—making it an essential target where resistance development might incur significant fitness costs. Second, while Complex I components share evolutionary conservation, sufficient structural differences exist between bacterial and human mitochondrial variants to potentially enable selective targeting. Third, A. baumannii's extensive antibiotic resistance profile necessitates novel targets outside conventional antibiotic classes.

The rational design approach for nuoA inhibitors would begin with detailed structural characterization through techniques like cryo-electron microscopy of the entire Complex I or modeling based on homologous structures. Computational screens could then identify compounds predicted to bind at functionally critical interfaces between nuoA and other Complex I subunits, potentially disrupting complex assembly or stability. Alternative strategies include designing compounds that mimic natural substrates or intermediates in electron transfer pathways, competitive inhibitors that bind at cofactor binding sites, or allosteric modulators that prevent conformational changes necessary for coupling electron transfer to proton pumping.

Methodologically, development would progress through biophysical screening assays measuring compound binding to recombinant nuoA, functional assays assessing inhibition of NADH oxidation activity , and whole-cell assays determining antimicrobial efficacy against A. baumannii. Particular attention must be paid to membrane permeability, as compounds must cross the bacterial outer membrane—a significant challenge for A. baumannii due to its naturally low permeability. Lead optimization would focus on enhancing selectivity over human Complex I, improving pharmacokinetic properties, and minimizing off-target effects.

The clinical potential for such inhibitors is particularly significant given A. baumannii's designation as a "red alert" human pathogen and the urgent need for novel antimicrobial approaches against multidrug-resistant strains. Success in this approach could provide valuable new therapeutic options against this challenging opportunistic pathogen.

What spectroscopic methods are most effective for analyzing electron transfer through recombinant nuoA-containing complexes?

Several spectroscopic methods provide complementary insights for analyzing electron transfer through recombinant nuoA-containing complexes, each with specific advantages for investigating different aspects of the process. UV-visible absorption spectroscopy offers straightforward quantitative monitoring of NADH oxidation by tracking the decrease in absorbance at 340 nm, where NADH absorbs strongly while NAD+ does not . This technique allows real-time kinetic measurements and is easily adapted for high-throughput screening. For more detailed mechanistic studies, stopped-flow spectroscopy can resolve rapid electron transfer events occurring in the millisecond range, critical for understanding the sequence of electron movements through the complex.

Electron Paramagnetic Resonance (EPR) spectroscopy provides detailed information about paramagnetic centers involved in electron transfer, including iron-sulfur clusters in Complex I. This technique can identify the redox states of these centers and track electrons as they move through the complex, offering insights into how nuoA mutations might affect electron flow. When combined with freeze-quench techniques, EPR can capture transient intermediates in the electron transfer process.

Fluorescence spectroscopy utilizing the intrinsic fluorescence of NADH provides an alternative approach for monitoring NADH oxidation with potentially greater sensitivity than absorption methods. The natural fluorescence decrease as NADH is converted to non-fluorescent NAD+ offers a direct readout of enzymatic activity. For more sophisticated analyses, Fluorescence Resonance Energy Transfer (FRET) approaches with strategically placed fluorophores can monitor conformational changes associated with electron transfer and proton pumping.

Resonance Raman spectroscopy provides unique insights into structural changes occurring during electron transfer by enhancing vibrational signals from chromophores involved in the process. This technique can detect subtle structural alterations in cofactors and amino acid residues participating in electron transfer, potentially revealing how nuoA mutations affect the electronic structure of the complex. Collectively, these spectroscopic approaches provide a comprehensive toolkit for investigating electron transfer through nuoA-containing complexes in A. baumannii, an opportunistic pathogen of increasing clinical concern .

How can researchers optimize detergent selection for maintaining nuoA structure and function during purification?

Optimizing detergent selection for maintaining nuoA structure and function during purification requires a systematic approach due to the critical impact of membrane mimetics on membrane protein stability. A comprehensive optimization strategy involves multiple phases of screening and validation. Initial broad-spectrum screening should evaluate detergents spanning different chemical classes and properties: maltoside detergents (DDM, UDM), glucoside detergents (OG), zwitterionic detergents (LDAO, FC-12), nonionic detergents (Triton X-100), and newer amphipathic polymers (SMALPs) or nanodiscs. This initial screen typically assesses protein extraction efficiency through Western blotting and basic quality assessment via size exclusion chromatography profiles.

For candidates showing promising extraction results, detailed stability assessment is essential. Thermal stability assays using differential scanning fluorimetry (DSF) or the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) can quantify protein stability in different detergents. These approaches provide thermal unfolding profiles that serve as valuable stability indicators. Functional validation is equally crucial, measuring NADH oxidation activity in different detergent environments to ensure preservation of catalytic function . Additionally, circular dichroism spectroscopy can verify that the protein maintains its expected secondary structure content.

For complex optimization, experimental design approaches can efficiently explore combinations of variables including detergent type, concentration, pH, salt concentration, and addition of specific lipids. The following table summarizes key considerations for major detergent classes:

Long-term storage stability must also be assessed, as many membrane proteins gradually lose activity even in optimized detergent conditions. Addition of specific lipids from A. baumannii membranes or glycerol as a stabilizing agent may improve long-term stability. This systematic approach ensures that recombinant nuoA maintains its structural and functional integrity throughout purification, enabling reliable downstream analyses of this important respiratory chain component from A. baumannii .

What strategies can minimize oxidative damage to nuoA during recombinant expression and purification?

Minimizing oxidative damage to nuoA during recombinant expression and purification requires a comprehensive strategy addressing vulnerability points throughout the experimental workflow. During expression, oxygen-limited growth conditions can be implemented by using lower shaking speeds (150-180 rpm) or specialized fermentation vessels with controlled oxygen levels. Expression host selection should consider strains with enhanced oxidative stress resistance, such as E. coli SHuffle or Origami strains with modified thioredoxin reductase and glutathione reductase systems. Media composition can be optimized by adding antioxidants like reduced glutathione (1-5 mM) or vitamin E (50-200 μM) to growth media, while inductively coupled plasma mass spectrometry (ICP-MS) can verify that trace metal levels are appropriate, as excess iron or copper can catalyze oxidative damage.

Cell disruption should employ gentle methods like osmotic shock or detergent-based lysis rather than high-pressure homogenization that can generate heat and reactive oxygen species. All buffers should be thoroughly degassed and supplemented with mild reducing agents like β-mercaptoethanol (5-10 mM) or dithiothreitol (1-5 mM), with care taken to avoid excessively strong reducing agents that might disrupt structural disulfide bonds. EDTA or EGTA (0.1-1 mM) should be included to chelate metal ions that catalyze oxidative reactions, while maintaining appropriate concentrations of physiologically relevant metals.

During purification, all steps should be performed at 4°C to slow oxidation kinetics, and fractions should be immediately analyzed for oxidative modifications using mass spectrometry to detect carbonylation, methionine oxidation, or cysteine modifications. Storage conditions should include oxygen-scavenging enzyme systems like glucose oxidase/catalase mixtures added to protein solutions prior to flash-freezing. For long-term storage, samples should be aliquoted into small volumes to minimize freeze-thaw cycles and stored under argon or nitrogen.

These strategies are particularly relevant for studying nuoA, which functions within the NADH-quinone oxidoreductase complex that naturally handles electron transfer and may contain redox-sensitive cofactors . Preserving nuoA in its native oxidation state is essential for accurate structural and functional studies that could provide insights into A. baumannii metabolism and potential therapeutic targeting .

How can researchers effectively apply genetic complementation assays to validate nuoA function in vivo?

Genetic complementation assays provide powerful tools for validating nuoA function in vivo, offering insights beyond in vitro biochemical approaches. A comprehensive complementation strategy begins with precise nuoA deletion strain construction using markerless deletion techniques to avoid polar effects on downstream genes. This creates a clean genetic background with a well-characterized phenotype, typically including growth defects under conditions requiring respiratory chain function, altered membrane potential, or modified susceptibility to respiratory chain inhibitors.

For effective complementation, expression vectors should be carefully designed with consideration of several factors. Plasmid copy number must be appropriate—while high-copy vectors may ensure sufficient expression, they can cause artifacts through protein overexpression. Low or medium-copy vectors often provide more physiologically relevant results. The promoter system should allow titratable expression, with options including native promoters for physiological expression levels or inducible promoters like tetracycline-responsive systems for controlled expression. Affinity tags should be strategically placed to minimize functional interference; C-terminal tags are often preferred for membrane proteins to avoid disrupting N-terminal membrane targeting sequences.

The complementation analysis should include multiple phenotypic assays to comprehensively evaluate functional restoration:

• Growth profiling under various carbon sources and oxygen conditions

• Membrane potential measurements using potential-sensitive dyes

• Direct Complex I activity assays in membrane preparations

• Antibiotic susceptibility profiling to assess changes in resistance patterns

• NADH/NAD+ ratio measurements to evaluate metabolic impact

To gain mechanistic insights, complementation with site-directed mutants is particularly valuable. Mutating conserved residues predicted to be involved in proton translocation or complex assembly can reveal structure-function relationships. Similarly, expressing orthologous nuoA genes from related species can identify species-specific functional aspects. Temporal control of complementation through inducible promoters allows researchers to distinguish between developmental versus ongoing requirements for nuoA function.

This comprehensive complementation approach provides robust validation of nuoA function in the physiologically relevant context of the living A. baumannii cell, complementing in vitro studies and providing insights into this important respiratory chain component of an increasingly significant opportunistic pathogen .